Luciferin used to study cell wall damage in single cells

The growing number of bacterial pathogens that have developed resistance to standard antibiotics has led researchers to look more closely at how antibiotics kill bacteria. A new technique uses chemiluminescent luciferins to allow scientists to observe the cell wall of a single bacterium rupture or, in some cases, to watch as the bacterium repairs its cell wall. The technique may prove useful to drug makers who are looking for new antibiotics and for novel ways to make older ones more effective.

The luciferin molecule creates chemiluminescent fluorescence when it combines with ATP luciferase. According to lead researcher Edward S. Yeung from the US Department of Energy’s Ames Laboratory at Iowa State University in Ames, luciferin, because of its size, is ideal for examining cell wall damage. At physiological pH, it becomes charged and will not pass through the cell wall. Reduce the pH, and the molecule becomes neutral and can pass quite easily. By controlling the pH, researchers can ensure that luciferin passes through only a cell wall that is damaged. Because of the molecule’s size, that damage can be small enough not to kill the bacterium outright.

Researchers at Ames Laboratory observed nonlethal damage to the cell wall of a single bacterium as well as cell lysis by using an optical microscope set up to collect chemiluminescence from bacteria expressing luciferase. By imaging the bacteria in a luciferin solution of neutral pH, even slight damage to the cell wall caused chemiluminescence (left) that was correlated to bright-field images (right). Courtesy of Edward S. Yeung.Yeung and his team genetically engineered a strain of Escherichia coli to produce ATP luciferase. Then, under carefully controlled conditions, they examined the luminescence response in a solution containing luciferin as the cell wall was ruptured or damaged by means of bacteriophages, dehydration or antibiotic attack.

Low-light imaging

The light levels emitted from a chemiluminescence event in a single bacterium are very low, so the group relied on an intensified CCD from Princeton Instruments Inc. of Trenton, N.J. According to Yeung, the more sensitive CCD, coupled with the ability to splice the gene, makes the technique possible. The rest of the system included a Nikon inverted microscope with a 100× objective, a complex electron-multiplying microchannel plate and a heated stage insert from World Precision Instruments Inc. of Sarasota, Fla. The researchers set up the experiment to capture bright-field and chemiluminescence images using a mechanical shutter from Edmund Optics Inc. of Barrington, N.J., to block light from the tungsten lamp they used for illumination. They set a 16-s imaging cycle that included a 10-s chemiluminescence image, a 2.5-s delay, a 5-ms bright-field image and a 3.495-s delay before taking another chemiluminescence image.

With the system, the researchers could clearly see bacteriophages destroying the bacteria. Some cells took less than 16 s to rupture — measured from the time the chemiluminescence began until it dissipated. Others took upward of 50 s. The dehydration and antibiotic tests showed the technique’s ability to measure smaller ruptures in the cell wall.

For the antibiotic experiments, the scientists rapidly exposed resistant bacteria to high levels of the antibiotic to which they are resistant. The bacteria showed an increase in chemiluminescence that lasted about 20 minutes and then a gradual decrease in luminescence lasting past the experiments’ end points.

Similarly, they observed that the transition of the cell membrane from liquid crystalline to a gel phase on dehydration led to leakage in the cell membrane. They allowed the bacteria to dehydrate for approximately 30 min and added more liquid to the solution. The chemiluminescence dropped sharply after complete dehydration but peaked again as the solution evaporated a second time.

Yeung said that increasing the dynamic range of the CCD camera will improve the images